Obesity and several other risk factors for atherosclerotic vascular disease (ASVD) including hypertension, dyslipidemia and type 2 diabetes are associated with insulin resistance (IR). It has been suspected, therefore, that IR is an important factor in the pathogenesis of ASVD although the nature of the connection between IR and ASVD has remained elusive. Free fatty acids (FFAs) have been established as a major link between obesity and IR based on evidence showing that most obese people have elevated plasma FFAs and that acute as well as chronic elevation of plasma FFAs cause IR. Recent data have shown that FFA induced IR was accompanied by intramyocellular (IMCL) accumulation of fat and diacylglycerol (DAG, a by-product of IMCL FFA reesterification to fat) by activation of protein kinase C (PKC) Beta II and delta (serine kinases known to be activated by DAG and to cause IR in rodents) and by a drastic (70%) reduction of IMCL IkappaB-alpha, (the inhibitor of the NFkappaB pathway which is known to be strongly pro-inflammatory and atherogenic). We propose to strengthen this putative link between FFA induced IR and ASVD by testing the following hypotheses: 1) that FFA induced activation of the serine kinases PKC beta II and delta and perhaps IkappaB kinase (IKK) in human muscle is associated with a decrease in insulin stimulated tyrosine phosphorylation of IRS-1 and of IRS-1 associated PI3 kinase; 2) that these changes precede the development of IR; 3) that the decrease in IkappaB-alpha results in activation of NFkappaB; 4) that PKC and IKK are involved in producing IR and activation of the IkappaB/NFkappaB pathway and 5) that the same mechanisms operative in healthy volunteers are also operative in patients with T2DM. We will test these hypotheses in normal and diabetic volunteers by performing euglycemic-hyperinsulinemic clamps with and without co-infusion of lipid plus heparin (to raise FFAs) and by obtaining serial muscle biopsies and blood samples. We believe that these studies will provide important new information relative to the mechanism by which obesity and FFAs cause IR and ASVD in human subjects.